Rm00021

The hypothalamus tissue samples were isolated and homogenized in ice-cold RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with proteinase inhibitors, and then subjected to centrifugation at 12,000 rpm for 15 min at 4°C to collect the supernatant. A BCA protein assay kit (P0012, Beyotime, Shanghai, China) was used to measure protein concentration. SDS-PAGE separated proteins were transferred onto PVDF membranes (Merck Millipore). The membranes were then blocked with tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% nonfat powdered milk for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies for AGRP (1:500, ab113481, Abcam), POMC (1:2,000, 66358-1-Ig, Proteintech), GHSR (1:500, ab85104, Abcam), or α-tubulin (1:4,000, AC007, ABclonal). After primary incubation, appropriate horseradish peroxidase (HRP)-labeled secondary antibodies (1:10,000, ABclonal) were incubated with membranes for 1 h at room temperature. Signal revelation was performed using an enhanced chemiluminescence substrate (RM00021, ABclonal) detection system. Band intensities were quantified using the ImageJ analysis program.

The in vitro-cultured SSCs were collected, and protein lysates (keygentec, Nangjing, China) were extracted at 4 °C for 30 min. The proteins were denatured in 5× SDS loading buffer at 100 °C for 5 min. The total cell proteins were resolved by 12% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% nonfat milk powder in TBST for 1 h, the PVDF membranes were incubated with polyclonal rabbit anti-Osm (1:1000, A6163, ABclonal, Woburn, MA, USA), monoclonal mouse anti-β actin (1:1000, sc-58673, Santa Cruz Biotechnology), polyclonal rabbit anti-Socs3 (1:1000, 14025-1-AP, Proteintech, Wuhan, China), monoclonal rabbit anti-p-Stat3 (1:1000, ab76315, Abcam), polyclonal rabbit anti-p-Akt (1:1000, AF0016, AFFinify) and polyclonal rabbit anti-GAPDH (1:1000, bs2188R, Bioss, Beijing, China) primary antibodies at 4 °C overnight. Then, horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit(1:20000, A21020-1, Abbkine) secondary antibodies were used at room temperature for 1 h, and the protein expression was detected by enhanced chemiluminescence (RM00021, ABclonal); gel imaging was performed with a ChemiDoc™ XRS+ (Bio-Rad, USA) with Image Lab™ software, and gray value analysis was performed with ImageJ.